Chronic alcohol intake synergistically increases the incidence of hepatocellular carcinoma (HCC) in hepatitis C patients, however, the underlying mechanisms remain poorly understood. A critical event in HCC is the deregulated expansion of tumor initiating cells (TICs), which are stem-like cells that promote treatment resistant HCC. The p53 tumor suppressor acts as a barrier against stem cell proliferation, and inactivation of p53 or its stabilizing partner NUMB leads to expansion of TICs. However, little is known about the control of the NUMB-p53 interaction and the mechanistic basis for p53 inactivation in TICs from HCC induced by alcohol and HCV. Ectopic induction of TLR4 in hepatocytes in HCV NsSa transgenic (Tg) mice generates TICs and liver tumors when the mice are chronically given alcohol. We demonstrated that stemness and tumor-initiating properties of TICs are dependent on TLR4-NANOG pathway and diminished levels of p53. The NUMB-p53 complex disintegrates upon NANOG-mediated activation of aPKCzeta, a NUMB kinase, leading to MDM2 ubiquitin ligase-dependent proteolysis of p53. Using affinity purification and tandem mass spectrometry, we have identified TBC1D15 as a NUMB-associated oncoprotein that can also disengage p53 from its protective association with NUMB, causing p53 degradation and deregulated TIC propagation. The expression of TBC1D15 is induced by the TLR4-NAN0G pathway and elevated in HCC in alcoholic HCV patients. We hypothesize TLR4-NAN0G-medlated NUMB phosphorylation and TBC1D15 upregulation coordinately trigger p53 degradation to promote self-renewal of TICs in liver oncogenesis caused by HCV and alcohol. To test this hypothesis, we will investigate whether: 1) NANOG-mediated induction of upstream AurA kinase and inhibition of the aPKC inhibitor LGL are responsible for activation of aPKCzeta and p53 degradation in TICs; 2) the newly disclosed aPKCzeta-NUMB and TBC1D15 pathways are mutually required for p53 degradation; 3) these pathways are evident and required for self-renewal of TICs isolated from HCC of alcoholic HCV patients; and 4) hepatocyte-specific expression of a non-phosphorylatable mutant of NUMB or hepatocyte-specific ablation of TbcldlS suppresses tumorigenesis in alcohol-fed HCV NsSaTg mice.